Simultaneous LC/ESI‐MS Separation Method for the Enantioseparation of Some New Anticonvulsant Drugs

A sensitive and specific method for the simultaneous determination of the enantiomeric purity of 2,6‐dimethylphenoxyacetyl derivatives as trans or cis racemic and enantiomeric forms with 2‐ or 4‐aminocyclohexanol moiety ( 1 , 2 , 3 , 6 ) and their amine analogs ( 8 , 9 ) was developed. The compounds studied are known for their anticonvulsant activity and the most interesting pharmacological results were those for (±)‐trans‐2‐(2,6‐dimethylphenoxy)‐N‐(2‐hydroxycyclohexyl)acetamide ( 1 ) as well as (±)‐trans‐2‐[(2,6‐dimethylphenoxy)ethyl]aminocyclohexanol ( 8 ). The analytical method for determining the enantiomeric purity of the compounds studied is based on direct separation of the analytes using a chiral stationary phase (Chiralpak AS column). The mass spectrometric analysis was done on a coupled liquid chromatograph–mass spectrometer system with an electrospray ionization source (LC/ESI‐MS). For the compounds 1 , 8 , and 9 , the method allows an excellent separation of enantiomers, with a resolution higher than 3.2, and a tailing factor of less than 1.67 with a final enantiomer purity better than 97.5%. Chirality 26:144–149, 2014. © 2014 Wiley Periodicals, Inc.     

Development of a Validated LC Method for Separation of Process‐Related Impurities Including the R‐enantiomer of S‐Pramipexole on Polysaccharide Chiral Stationary Phases

Despite the availability of a few methods for individual separation of S‐pramipexole from its process‐related impurities, no common liquid chromatography (LC) method is reported so far in the literature. The present article describes the development of a single‐run LC method for simultaneous determination of S‐pramipexole and its enantiomeric and process‐related impurities on a Chiralpak AD‐H (150 x 4.6 mm, 5μm) column using n‐hexane/ethanol/n‐butylamine (75:25:0.1 v/v/v) as a mobile phase in an isocratic mode of elution at a flow rate of 1.2 ml/min at 30°C. The chromatographic eluents were monitored at a wavelength of 260 nm using a photodiode array detector. Excellent enantioseparation with good resolutions (Rs ≥ 2.88) and peak shapes (A_s ≤ 1.21) for all analytes was achieved. The proposed method was validated according to International Conference Harmonization (ICH) guidelines in terms of accuracy, precision, sensitivity, and linearity. Limits of quantification of impurities (0.25–0.55 μg/ml) indicate the highest sensitivity achievable by the proposed method. The method has an advantage of selectivity and suitability for routine determination of not only chiral impurity but also all possible related substances in active pharmaceutical ingredients of S‐pramipexole. Chirality 27:430–435, 2015. © 2015 Wiley Periodicals, Inc.     

Preparation and Studies of Chiral Stationary Phases Containing Enantiopure Acridino‐18‐Crown‐6 Ether Selectors

The enantiomeric separation ability of the newly prepared chiral stationary phases containing acridino‐18‐crown‐6 ether selectors was studied by high‐performance liquid chromatography (HPLC). The chiral stationary phases separated the enantiomers of selected protonated primary aralkylamines efficiently. The best results were found for the separation of the mixtures of enantiomers of NO₂‐PEA. Chirality 26:651–654, 2014. © 2014 Wiley Periodicals, Inc.     

Enantiomeric Separation of Underivatized Amino Acids: Predictability of Chiral Recognition on Ristocetin A Chiral Stationary Phase

The present work aimed to investigate the predictability of the chromatographic behavior for the separation of underivatized amino acids on ristocetin A, known as Chirobiotic R, using a DryLab high‐performance liquid chromatography (HPLC) method development software, which is typically used to predict the effect of changing various chromatographic parameters on resolution in the reversed phase mode. After implementing the basic runs, and judging the predictability via the computed resolution map, it can be deduced that the chiral recognition mechanisms tend towards a hydrophilic interaction chromatography rather than the reversed phase mode, which limits the ability of DryLab software to predict separations on Chirobiotic R. Chirality 26:132–135, 2014. © 2014 Wiley Periodicals, Inc.     

Screening Approach for Chiral Separation of β‐Aminoketones by HPLC on Various Polysaccharide‐Based Chiral Stationary Phases

Nine β‐aminoketones were synthesized via Mannich reaction when benzaldehyde was condensed with some primary amines and acetophenone. The purified compounds were identified by using spectroscopic methods. The enantiomeric separation of these derivatives was carried out by high‐performance liquid chromatography (HPLC) using several coated and immobilized polysaccharide stationary phases, namely, Chiralcel^® OD‐H, Chiralcel^® OD, Chiralcel^® OJ, Chiralpak^® AD, Chiralpak^® IA, and Chiralpak^® IB using different mobile phases composed of n‐hexane and alcohol mixed in various ratios or pure ethanol or isopropanol. The retention behavior and selectivity of these chiral stationary phases were examined in isocratic normal phase mode. The results indicate that cellulose derivatives have higher enantioselectivity than amylose derivatives for the separation of racemic β‐amino ketones. Chirality 27:332–338, 2015. © 2015 Wiley Periodicals, Inc.     

Enantioresolution of Chiral Derivatives of Xanthones on (S,S)‐Whelk‐O1 and l‐Phenylglycine Stationary Phases and Chiral Recognition Mechanism by Docking Approach for (S,S)‐Whelk‐O1

The resolution of seven enantiomeric pairs of chiral derivatives of xanthones (CDXs) on (S,S)‐Whelk‐O1 and l ‐phenylglycine chiral stationary phases (CSPs) was systematically investigated using multimodal elution conditions (normal‐phase, polar‐organic, and reversed‐phase). The (S,S)‐Whelk‐O1 CSP, under polar‐organic conditions, demonstrated a very good power of resolution for the CDXs possessing an aromatic moiety linked to the stereogenic center with separation factor and resolution factor ranging from 1.91 to 7.55 and from 6.71 to 24.16, respectively. The chiral recognition mechanisms were also investigated for (S,S)‐Whelk‐O1 CSP by molecular docking technique. Data regarding the CSP–CDX molecular conformations and interactions were retrieved. These results were in accordance with the experimental chromatographic parameters regarding enantioselectivity and enantiomer elution order. The results of the present study fulfilled the initial objectives of enantioselective studies of CDXs and elucidation of intermolecular CSP–CDX interactions. Chirality 25:89–100, 2013. © 2012 Wiley Periodicals, Inc.     

Resolution,determination of enantiomeric purity and chiral recognition mechanism of new xanthone derivatives on (S,S)‐whelk‐O1 stationary phase

The enantioresolution and determination of the enantiomeric purity of 32 new xanthone derivatives, synthesized in enantiomerically pure form, were investigated on (S ,S )‐Whelk‐O1 chiral stationary phase (CSP). Enantioselectivity and resolution (α and R_S) with values ranging from 1.41–6.25 and from 1.29–17.20, respectively, were achieved. The elution was in polar organic mode with acetonitrile/methanol (50:50 v/v ) as mobile phase and, generally, the (R )‐enantiomer was the first to elute. The enantiomeric excess (ee ) for all synthesized xanthone derivatives was higher than 99%. All the enantiomeric pairs were enantioseparated, even those without an aromatic moiety linked to the stereogenic center. Computational studies for molecular docking were carried out to perform a qualitative analysis of the enantioresolution and to explore the chiral recognition mechanisms. The in silico results were consistent with the chromatographic parameters and elution orders. The interactions between the CSP and the xanthone derivatives involved in the chromatographic enantioseparation were elucidated.     

Superficially Porous Particle Based Hydroxypropyl‐β‐cyclodextrin Stationary Phase for High‐Efficiency Enantiomeric Separations

A superficially porous particle (SPP)‐based hydroxypropyl‐β‐cyclodextrin (HPBCD) chiral stationary phase (CSP) was produced and its chromatographic performance was compared to both 5 µm and 3 µm fully porous particle (FPP)‐based CSPs. The relative surface coverage of the HPBCD chiral selector on each particle was approximately equal, which resulted in equivalent enantiomeric selectivity (α) values on each phase when constant mobile phase conditions were used. Under such conditions, the SPP column resulted in greatly reduced analysis times and three times greater efficiencies compared to the FPP columns. When higher flow rates were used, efficiency gains per analysis times were five times greater for the SPP column compared to the FPP‐based columns. When the mobile phases were altered to give similar analysis times on each column, resolution values were doubled for the SPP column. Finally, the novel SPP based HPBCD column proved to be stable for 500 injections under high flow rate (4.5 mL/min) and high pressure (400 bar) conditions used for an ultrafast (~45 sec) enantiomeric separation. Chirality 27:788–794, 2015. © 2015 Wiley Periodicals, Inc.     

Chiral Separation of Uncharged Pomalidomide Enantiomers Using Carboxymethyl‐β‐Cyclodextrin: A Validated Capillary Electrophoretic Method

The racemic mixture of pomalidomide (POM), a second‐generation immunomodulatory uncharged drug, was separated into enantiomers by capillary zone electrophoresis for the first time. Seven different chargeable cyclodextrin (CD) derivatives were screened as complexing agents and chiral selectors, investigating the stability of the POM‐CD inclusion complexes and their enantiodiscriminating capacities. Based on preliminary experiments, carboxymethyl‐β‐CD (CM‐β‐CD) was found to be the most effective chiral selector. Factors influencing enantioseparation were systematically optimized, using an orthogonal experimental design. Optimal parameters (background electrolyte [BGE]: 50 mM Tris‐acetate buffer, pH 6.5, containing 15 mM CM‐β‐CD; capillary temperature: 20°C; voltage applied +15 kV) allowed baseline separation of POM enantiomers with a resolution as high as 4.87. The developed method was validated, in terms of sensitivity (limit of detection and limit of quantification), linearity, accuracy, repeatability, and intermediate precision. Chirality 28:199–203, 2016. © 2015 Wiley Periodicals, Inc.     

Enantiomeric NMR signal separation behavior and mechanism of samarium(III) and neodymium(III) complexes with (S,S)‐ethylenediamine‐N,N'‐disuccinate

Enantiomeric ¹H and ¹³C NMR signal separation behaviors of various α‐amino acids and DL‐tartarate were investigated by using the samarium(III) and neodymium(III) complexes with (S ,S )‐ethylenediamine‐N ,N' ‐disuccinate as chiral shift reagents. A relatively smaller concentration ratio of the lanthanide(III) complex to substrates was suitable for the neodymium(III) complex compared with the samarium(III) one, striking a balance between relatively greater signal separation and broadening. To clarify the difference in the signal separation behavior, the chemical shifts of β‐protons for fully bound D‐ and L‐alanine (δ_b(D) and δ_b(L)) and their adduct formation constants (K s) were obtained for both metal complexes. Preference for D‐alanine was similarly observed for both complexes, while it was revealed that the difference between the δ_b(D) and δ_b(L) values is the significant factor to determine the enantiomeric signal separation. The neodymium(III) and samarium(III) complexes can be used complementarily for higher and smaller concentration ranges of substrates, respectively, because the neodymium(III) complex gives the larger difference between the δ_b(D) and δ_b(L) values with greater signal broadening compared to the samarium(III) complex.     

Enantiomeric Separation and Simulation Studies of Pheniramine,Oxybutynin, Cetirizine,and Brinzolamide Chiral Drugs on Amylose‐Based Columns

Solid phase extraction ( SPE)‐chiral separation of the important drugs pheniramine, oxybutynin, cetirizine, and brinzolamide was achieved on the C₁₈ cartridge and AmyCoat (150 x 46 mm) and Chiralpak AD (25 cm x 0.46 cm id) chiral columns in human plasma. Pheniramine, oxybutynin, cetirizine, and brinzolamide were resolved using n‐hexane‐2‐PrOH‐DEA (85:15:0.1, v/v), n‐hexane‐2‐PrOH‐DEA (80:20:0.1, v/v), n‐hexane‐2‐PrOH‐DEA (70:30:0.2, v/v), and n‐hexane‐2‐propanol (90:10, v/v) as mobile phases. The separation was carried out at 25 ± 1 ºC temperature with detection at 225 nm for cetirizine and oxybutynin and 220 nm for pheniramine and brinzolamide. The flow rates of the mobile phases were 0.5 mLmin^‐1. The retention factors of pheniramine, oxybutynin, cetirizine and brinzolamide were 3.25 and 4.34, 4.76 and 5.64, 6.10 and 6.60, and 1.64 and 2.01, respectively. The separation factors of these drugs were 1.33, 1.18, 1.09 and 1.20 while their resolutions factors were 1.09, 1.45, 1.63 and 1.25, and 1.15, respectively. The absolute configurations of the eluted enantiomers of the reported drugs were determined by simulation studies. It was observed that the order of enantiomers elution of the reported drugs was S‐pheniramine > R‐pheniramine; R‐oxybutynin > S‐oxybutynin; S‐cetirizine > R‐cetirizine; and S‐brinzolamide > R‐brinzolamide. The mechanism of separation was also determined at the supramolecular level by considering interactions and modeling results. The reported SPE‐chiral high‐performance liquid chromatography ( HPLC) methods are suitable for the enantiomeric analyses of these drugs in any biological sample. In addition, simulation studies may be used to determine the absolute configuration of the first and second eluted enantiomers. Chirality 26:136–143, 2014. © 2014 Wiley Periodicals, Inc.     

GABAA agonists: Resolution and pharmacology of (+)- and (−)-isoguvacine oxide

(3SR,4RS)-3,4-Epoxypiperidine-4-carboxylic acid (isoguvacine oxide) is a potent and specific GABA_A receptor agonist. Isoguvacine oxide, originally designed as a potentially alkylating agonist, turned out to interact with the GABA_A receptor in a fully reversible manner. The protected form of isoguvacine oxide, benzyl (3SR,4RS)-1-(benzyloxycarbonyl)-3,4-epoxypiperidine-4-carboxylate ( 1 ) (Scheme 1), has now been resolved by chiral chromatography using cellulose triacetate as a chiral stationary phase. The enantiomers of 1 (ee ≥ 98.8%) were subsequently deprotected by hydrogenolysis. Whereas both enantiomers of isoguvacine oxide were inactive as inhibitors of the binding of [³H]GABA to GABA_B receptor sites (IC₅₀ > 100 μM), (+)-isoguvacine oxide (IC₅₀ = 0.20 ± 0.03 μM) and (?)-isoguvacine oxide (IC₅₀ = 0.32 ± 0.05 μM) showed comparable potencies as inhibitors of the binding of [³H]GABA to GABA_A receptor sites. Furthermore, (+)-isoguvacine oxide (EC₅₀ = 6 μM; 33% relative efficacy) and (?)-isoguvacine oxide (EC₅₀ = 5 μM; 38% efficacy relative to 10 μM muscimol) were approximately equipotent and equiefficacious as stimulators of the binding of [³H]diazepam to the GABA_A receptor-associated benzodiazepine site. This latter effect is an in vitro estimate of GABA_A agonist efficacy. These pharmacological data for isoguvacine oxide and its enantiomers do not seem to support our earlier conception of the topography of the GABA_A recognition site(s), derived from extensive structure—activity studies on GABA_A agonists. Thus, the model of the GABA_A recognition site(s) comprising a narrow cleft or pocket, in which the anionic moiety of the zwitterionic GABA_A agonists is assumed to be embedded during receptor activation, may have to be revised. © 1995 Wiley-Liss, Inc.     

Chiral Amine Enantiomeric Excess Determination Using Self‐Assembled Octahedral Fe(II)‐Imine Complexes

A receptor assembly composed of iron(II) triflate and pyridine‐2,6‐dicarbaldehyde was used to determine the enantiomeric excess (ee) of alpha‐chiral primary amines using circular dichroism spectroscopy. The alpha chiral amines condense with the dialdehyde to form a diimine, which forms a 2:1 octahedral complex with iron(II). The ee values of unknown concentrations of alpha‐chiral amines were determined by constructing calibration curves for each amine and then measuring the ellipticity at 600 nm. This improves our previously reported assay for ee determination of chiral primary amines by further increasing the wavelength at which CD is measured and reducing the absolute error of the assay. Chirality 27:294–298, 2015. 2015 Wiley Periodicals, Inc.     

Enantiomeric Separation of Bicyclo[2.2.2]octane‐Based 2‐Amino‐3‐Carboxylic Acids on Macrocyclic Glycopeptide Chiral Stationary Phases

Direct high‐performance liquid chromatographic (HPLC) separation of four bicyclo[2.2.2]octane based 2‐amino‐3‐carboxylic acid enantiomers were developed on chiral stationary phases (CSPs) containing different macrocyclic glycopeptide antibiotic selectors. The analyses were performed under reversed‐phase, polar organic and polar ionic mode on macrocyclic‐glycopeptide‐based Chirobiotic T, T2, TAG, and R columns. The effects of the mobile phase composition including the acid and base modifier, the structure of the analytes, and the temperature on the separations were investigated. Experiments were achieved at constant mobile phase compositions on different stationary phases in the temperature range 5–40°C. Thermodynamic parameters were calculated from plots of ln k or ln α versus 1/T. It was recognized that the enantioseparations in reversed‐phase and polar organic mode were enthalpically driven, but under polar‐ionic conditions entropically driven enantioseparation was observed as well. Baseline separation and determination of elution sequence were achieved in all cases. Chirality 26:200–208, 2014. © 2014 Wiley Periodicals, Inc.     

Chiral Recognition of N‐Phthaloyl,N‐Tetrachlorophthaloyl,and N‐Naphthaloyl α‐Amino Acids and Their Esters on Polysaccharide‐Derived Chiral Stationary Phases

Enantiomeric separations of N‐phthaloyl (N‐PHT), N‐tetrachlorophthaloyl (N‐TCPHT), and N‐naphthaloyl (N‐NPHT) α‐amino acids and their esters were examined on several kinds of polysaccharide‐derived chiral stationary phases (CSPs). Resolution capability of CSPs was greater Chiralcel OF than the others for N‐PHT and N‐NPHT α‐amino acids and their esters. In N‐TCPHT α‐amino acids and their esters, good enantioselectivities showed Chiralcel OG for N‐TCPHT α‐amino acids, Chiralpak AD for N‐TCPHT α‐amino acid methyl esters, and Chiralcel OD for N‐TCPHT α‐amino acid ethyl esters, respectively. From the results of liquid chromatography and computational chemistry, it is concluded that l ‐form is preferred and more retained with electrostatic interaction in case of interaction between N‐PHT α‐amino acid derivatives and Chiralcel OF, N‐TCPHT α‐amino acid derivatives and Chiralcel OD, and N‐NPHT α‐amino acid derivatives and Chiracel OF. On the other hand, d ‐form is preferred and more retained with van der Waals interaction in case of interaction between N‐TCPHT α‐amino acid ester derivatives and Chiralcel OG and Chiralpak AD. Chirality 24:1037–1046, 2012. © 2012 Wiley Periodicals, Inc.     

Synthesis and Application of C2 and C3 Symmetric (R)‐Phenylglycinol‐Derived Chiral Stationary Phases

A C3 symmetric (R)‐phenylglycinol N‐1,3,5‐benzenetricarboxylic acid‐derived chiral stationary phase (CSP) and three C2 symmetric (R)‐phenylglycinol CSPs were newly synthesized using o‐, m‐, and p‐phthaloyl dichlorides. © 2016 Wiley Periodicals, Inc. These CSPs were used to compare the resolution of 25 chiral samples using a previously reported 3,5‐dinitrobenzoyl (R)‐phenylglycinol‐derived CSP. Even though all CSPs have the same chiral moiety, the C3 symmetric CSP showed the best resolution. Chirality 28:186–191, 2016.© 2016 Wiley Periodicals, Inc.     

High‐Performance Liquid Chromatographic Enantioseparation of Cyclic β‐Amino Acids on Zwitterionic Chiral Stationary Phases Based on Cinchona Alkaloids

Stereoselective high‐performance liquid chromatographic separations of eight sterically constrained cyclic β‐amino acid enantiomer pairs were carried out using the newly developed Cinchona alkaloid‐based zwitterionic chiral stationary phases Chiralpak ZWIX(+) and ZWIX(?). The effects of the mobile phase composition, the nature and concentrations of the acid and base additives, the counterions and temperature on the separations were investigated. The changes in standard enthalpy, Δ(ΔH°), entropy, Δ(ΔS°), and free energy, Δ(ΔG°), were calculated from the linear van't Hoff plots derived from the ln α vs. 1/T curves in the studied temperature range (10–50°C). The values of the thermodynamic parameters depended on the nature of the selectors and the structures of the analytes. Unusual temperature behavior was observed on the ZWIX(?) column: decreased retention times were accompanied by increased separation factors with increasing temperature. On the ZWIX(+) column only enthalpically, whereas on the ZWIX(?) column both enthalpically and entropically driven separations were observed. The elution sequence was determined in all cases and was observed to be the opposite on ZWIX(+) and on ZWIX(?). Chirality 27:563–570, 2015. © 2015 Wiley Periodicals, Inc.     

Chiral Ligand‐Exchange Resolution of Underivatized Amino Acids on a Dynamically Modified Stationary Phase for RP‐HPTLC

The synthesis of Spi(τ‐dec), derived from the selective alkylation of L‐spinacine (4,5,6,7‐tetrahydro‐1H‐imidazo[4,5‐c]pyridine‐6‐carboxylic acid) at the τ‐nitrogen of its heteroaromatic ring, with a linear hydrocarbon chain of 10 carbon atoms, is described here for the first time. Spi(τ‐dec) was successfully employed in the past to prepare home‐made chiral columns for chiral ligand‐exchange high‐performance liquid chromatography. In the present article a new method is described, using Spi(τ‐dec) as a chiral selector in high‐performance thin‐layer chromatography (HPTLC): commercial hydrophobic plates were first coated with Spi(τ‐dec) and then treated with copper sulfate. The performance of this new chiral stationary phase was tested against racemic mixtures of aromatic amino acids, after appropriate optimization of both the conditions of preparation of the plates and the mobile phase composition. The enantioselectivity values obtained for the studied compounds were higher than those reported in the literature for similar systems. The method employed here for the preparation of chiral HPTLC plates proved practical, efficient, and inexpensive. Chirality 26:313–318, 2014. © 2014 Wiley Periodicals, Inc.     

HPLC‐Fluorescence Method for the Enantioselective Analysis of Propranolol in Rat Serum Using Immobilized Polysaccharide‐Based Chiral Stationary Phase

A stereoselective high‐performance liquid chromatographic (HPLC) method was developed and validated to determine S‐(?)‐ and R‐(+)‐propranolol in rat serum. Enantiomeric resolution was achieved on cellulose tris(3,5‐dimethylphenylcarbamate) immobilized onto spherical porous silica chiral stationary phase (CSP) known as Chiralpak IB. A simple analytical method was validated using a mobile phase consisted of n‐hexane‐ethanol‐triethylamine (95:5:0.4%, v/v/v) at a flow rate of 0.6 mL min^‐1 and fluorescence detection set at excitation/emission wavelengths 290/375 nm. The calibration curves were linear over the range of 10–400 ng mL^‐1 (R = 0.999) for each enantiomer with a detection limit of 3 ng mL^‐1. The proposed method was validated in compliance with ICH guidelines in terms of linearity, accuracy, precision, limits of detection and quantitation, and other aspects of analytical validation. Actual quantification could be made for propranolol isomers in serum obtained from rats that had been intraperitoneally (i.p.) administered a single dose of the drug. The proposed method established in this study is simple and sensitive enough to be adopted in the fields of clinical and forensic toxicology. Molecular modeling studies including energy minimization and docking studies were first performed to illustrate the mechanism by which the active enantiomer binds to the β‐adrenergic receptor and second to find a suitable interpretation of how both enantiomers are interacting with cellulose tris(3,5‐dimethylphenylcarbamate) CSP during the process of resolution. The latter interaction was demonstrated by calculating the binding affinities and interaction distances between propranolol enantiomers and chiral selector. Chirality 26:194–199, 2014. © 2014 Wiley Periodicals, Inc.